Compositions and methods for diagnosing and treating mood disorders

ABSTRACT

The present invention provides methods for diagnosing mental disorders such as mood disorders, including bipolar disorder I and II and major depression. The invention also provides methods of identifying modulators of such mental disorders as well as methods of using these modulators to treat patients suffering from such mental disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. S No.60/423,247, filed Nov. 1, 2002 and U.S. S No. 60/431,454, filed Dec. 6,2002, the disclosures of which are hereby incorporated by reference intheir entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] Clinical depression, including both bipolar disorders and majordepression disorders, is a major public health problem, affecting anestimated 9.5% of the adult population of the United States each year.While it has been hypothesized that mental disorders, including mooddisorders such as major depression and bipolar disorder as well aspsychotic disorders such as schizophrenia, have complex genetic roots,little progress has been made in identifying gene sequences and geneproducts that play a role in causing these disorders, as is true formany diseases with a complex genetic origin (see, e.g., Burmeister,Biol. Psychiatry 45:522-532 (1999)). Relying on the discovery thatcertain genes expressed in particular brain pathways and regions arelikely involved in the development of mental disorders, the presentinvention provides methods for diagnosis and treatment of mentaldisorders, as well as methods for identifying compounds effective intreating mental disorders.

BRIEF SUMMARY OF THE INVENTION

[0004] In order to further understand the neurobiology of mood disorderssuch as bipolar disorders (BP) and major depression disorders (MDD), theinventors of the present application have used DNA microarrays to studyexpression profiles of human post-mortem brains from patients diagnosedwith BP or MDD. The work has focused on three brain regions: theanterior cingulated cortex (AnCg), the dorsolateral prefrontal cortex(DLPFC), and the cerebellum (CB).

[0005] The present invention demonstrates, for the first time,differential expression of the 72 nucleic acids listed in Table 2, the16 nucleic acids listed in Table 3, or the 967 nucleic acids listed inTable 4, in the brains of patients suffering from mood disorders, suchas bipolar disorder and major depression disorder, in comparison withnormal control subjects. In addition, the present invention identifiesbiochemical pathways involved in mood disorders, where the proteinsencoded by the nucleic acids listed in Table 2, 3, or 4 are componentsof the biochemical pathways (e.g., the bFGF signal transduction pathway,the GPCR and cAMP/PI/Rho pathways, the proteasome pathway, the oxidativephosphorylation pathway, Myelination, Cytochrome P450, or the GABA andglutamate pathways; see also FIGS. 1-5, 10-13, and 15).

[0006] Finally, genes that are differentially expressed in MDD or BP andby gender are useful in diagnosing mood disorders, as the prevalence ofcertain mood disorders shows a gender bias. Differential expression bybrain region similarly is a useful diagnostic and therapeutic tool, ascertain mood disorders primarily affect certain brain regions.

[0007] This invention thus provides methods for determining whether asubject has or is predisposed for a mental disorder such as bipolardisorder or major depression disorder. The invention also providesmethods of providing a prognosis and for monitoring disease progressionand treatment. Furthermore, the present invention provides nucleic acidand protein targets for assays for drugs for the treatment of mentaldisorders such as bipolar disorder and major depression disorder.

[0008] In some embodiments, the methods comprise the steps of: (i)obtaining a biological sample from a subject; (ii) contacting the samplewith a reagent that selectively associates with a polynucleotide orpolypeptide encoded by a nucleic acid that hybridizes under stringentconditions to a nucleotide sequence listed in Table 2, 3 or 4; and (iii)detecting the level of reagent that selectively associates with thesample, thereby determining whether the subject has or is predisposedfor a mental disorder.

[0009] In some embodiments, the reagent is an antibody. In someembodiments, the reagent is a nucleic acid. In some embodiments, thereagent associates with a polynucleotide. In some embodiments, thereagent associates with a polypeptide. In some embodiments, thepolynucleotide comprises a nucleotide sequence of a gene listed in Table2, 3, or 4. In some embodiment, the polypeptide comprises an amino acidsequence of a gene listed in Table 2, 3, or 4. In some embodiments, thelevel of reagent that associates with the sample is different (i.e.,higher or lower) from a level associated with humans without a mentaldisorder. In some embodiments, the biological sample is obtained fromamniotic fluid. In some embodiments, the mental disorder is a mooddisorder. In some embodiments, the mood disorder is selected from thegroup consisting of bipolar disorder and major depression disorder.

[0010] The invention also provides methods of identifying a compound fortreatment of a mental disorder. In some embodiments, the methodscomprises the steps of: (i) contacting the compound with a polypeptide,which is encoded by a polynucleotide that hybridizes under stringentconditions to a nucleic acid comprising a nucleotide sequence of Table2, 3, or 4; and (ii) determining the functional effect of the compoundupon the polypeptide, thereby identifying a compound for treatment of amental disorder.

[0011] In some embodiments, the contacting step is performed in vitro.In some embodiment, the polypeptide comprises an amino acid sequence ofa gene listed in Table 2, 3, or 4. In some embodiments, the polypeptideis expressed in a cell or biological sample, and the cell or biologicalsample is contacted with the compound. In some embodiments, the mentaldisorder is a mood disorder or psychotic disorder. In some embodiments,the mood disorder is selected from the group consisting of bipolardisorder I and II and major depression. In some embodiments, thepsychotic disorder is schizophrenia. In some embodiments, the methodsfurther comprise administering the compound to an animal, e.g., ananimal subjected to stress as a model for depression and determining theeffect on the animal, e.g., an invertebrate, a vertebrate, or a mammal.In some embodiments, the determining step comprises testing the animal'smental function.

[0012] In some embodiments, the methods comprise the steps of (i)contacting the compound to a cell, the cell comprising a polynucleotidethat hybridizes under stringent conditions to a nucleotide sequence ofTable 2, 3, or 4; and (ii) selecting a compound that modulatesexpression of the polynucleotide, thereby identifying a compound fortreatment of a mental disorder. In some embodiments, the polynucleotidecomprises a nucleotide sequence listed in Table 2, 3, or 4. In someembodiment, the expression of the polynucleotide is enhanced. In someembodiments, the expression of the polynucleotide is decreased. In someembodiments, the methods further comprise administering the compound toan animal and determining the effect on the animal. In some embodiments,the determining step comprises testing the animal's mental function. Insome embodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the mood disorder is selected from thegroup consisting of bipolar disorder I and II and major depression. Insome embodiments, the psychotic disorder is schizophrenia.

[0013] The invention also provides methods of treating a mental disorderin a subject. In some embodiments, the methods comprise the step ofadministering to the subject a therapeutically effective amount of acompound identified using the methods described above. In someembodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the mood disorder is selected from thegroup consisting of bipolar disorder I and II and major depression. Insome embodiments, the psychotic disorder is schizophrenia. In someembodiments, the compound is a small organic molecule, an antibody, anantisense molecule, aptamer, or a peptide.

[0014] The invention also provides methods of treating mental disordersin a subject, comprising the step of administering to the subject atherapeutically effective amount of a polypeptide, which is encoded by apolypeptide that hybridizes under stringent conditions to a nucleic acidof Table 2, 3, or 4. In some embodiments, the polypeptide comprises anamino acid sequence encoded by a gene listed in Table 2, 3, or 4. Insome embodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the psychotic disorder is schizophrenia.In some embodiments, the mood disorder is a bipolar disorder or majordepression.

[0015] The invention also provides methods of treating mental disordersin a subject, comprising the step of administering to the subject atherapeutically effective amount of a polypeptide, wherein thepolypeptide hybridizes under stringent conditions to a nucleic acid ofTable 2, 3, or 4. In some embodiments, the mental disorder is a mooddisorder or psychotic disorder. In some embodiments, the psychoticdisorder is schizophrenia. In some embodiments, the mood disorder is abipolar disorder or major depression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Table 1: Table 1 lists genes differentially expressed in mooddisorder subjects.

[0017] Table 2: Table 2 lists 72 genes differentially expressed in mooddisorder subjects.

[0018] Table 3: Table 3 lists 16 genes differentially expressed inspecific brain regions and mood disorder.

[0019] Table 4: Table 4 lists 967 genes differentially expressed in mooddisorder subjects as determined by microarray analysis. Flag 1 indicatesthat the differential expression of the gene was confirmed by Real timePCR. Flag 2 indicates that differential expression of the gene wasconfirmed by anti-depressant studies. Flag 3 indicates that the genebelongs to an enriched gene ontology. Up and down indicates thedirection of the changes compared to controls.

[0020] Table 5: Table 5 lists Real time PCR results on sample genes thatare differentially expressed in mood disorder subjects.

[0021] Table 6: Table 6 lists anti-depressant treatment results forgenes that are differentially expressed in mood disorder subjects.

[0022] Table 7: Tables 7A-D lists the gene ontology of selected genesdifferentially expressed in mood disorder subjects.

[0023] Table 8: Table 8 lists sample of genes that are differentiallyexpressed in mood disorder subjects and are potential druggable targets.

[0024]FIG. 1: FIG. 1 shows selected biochemical pathways for genesdifferentially expressed in mood disorder subjects.

[0025]FIG. 2: FIG. 2 summarizes functions for signal transductiontranscripts differentially expressed in MDD subjects.

[0026]FIG. 3: FIG. 3 shows bFGF pathway transcripts differentiallyexpressed in MDD subjects.

[0027]FIG. 4: FIG. 4 shows values for differential expression of bFGFtranscripts in MDD subjects.

[0028]FIG. 5: FIG. 5 shows selected biochemical pathways that aredysregulated in mood disorders.

[0029]FIG. 6: FIG. 6 shows selected biochemical pathways that aredysregulated in BP subjects.

[0030]FIG. 7: FIG. 7 shows three genes overexpressed in mood disordersubjects that are located in the same chromosomal region.

[0031]FIG. 8: FIG. 8 shows three genes overexpressed in mood disordersubjects that are located on 15q11-13 in the Prader-Willi region.

[0032]FIG. 9: FIG. 9 shows certain genes regulated in human postmortemtissue and by antidepressants in rats.

[0033]FIG. 10: FIG. 10 shows selected biochemical pathways (i.e., theGPCR and cAMP/PI/Rho pathways) for genes differentially expressed inmood disorder subjects. Two G protein coupled receptors, GPR37 andGPRC5B, are increased in both AnCg and DLPFC of BP patients, anddecreased in MD. As downstream signaling pathways of GPCR, genesinvolved in cAMP pathway signaling are increased n BP patients, anddecreased in MD. Genes involved in phosphatidylinositol pathways arederegulated specifically in MD.

[0034]FIG. 11: FIG. 11 shows a selected biochemical pathway (i.e., theproteasome pathway) for genes differentially expressed in mood disordersubjects. The proteasome is an assembly of 28 alpha and beta subunitsthat functions to degrade proteins. The proteasome is involved inregulation of protein turnover and in particular oxidized proteins.There is an over representation of proteasome genes found in corticalregions of BP, but not in the cerebellum, suggesting that somefunctional compensation in the proteasome is occurring in BP patients.

[0035]FIG. 12: FIG. 12 shows a selected biochemical pathway (i.e., theoxidative phosphorylation pathway) for genes differentially expressed inmood disorder subjects. The oxidative phosphorylation classification isinvolved in bioenergetics, metabolism, and as a byproduct can producereactive oxygen species. This pathway is overly expressed in bothbipolar and major depression, with differences between cortical regionsand cerebellum.

[0036]FIG. 13: FIG. 13 shows an example of a growth factor system (e.g.,FGF) that is altered in mood disorders.

[0037]FIG. 14: FIG. 14 shows RealTime PCR results which confirm thatselected FGF-related genes first identified using microarray analysisare differentially expressed in mood disorders.

[0038]FIG. 15: FIG. 15 shows selected genes in biochemical pathwaysinvolving GABA and glutamate that are differentially expressed in mooddisorder subjects.

DEFINITIONS

[0039] A “mental disorder” or “mental illness” or “mental disease” or“psychiatric or neuropsychiatric disease or illness or disorder” refersto mood disorders (e.g., major depression, mania, and bipolardisorders), psychotic disorders (e.g., schizophrenia, schizoaffectivedisorder, schizophreniform disorder, delusional disorder, briefpsychotic disorder, and shared psychotic disorder), personalitydisorders, anxiety disorders (e.g., obsessive-compulsive disorder) aswell as other mental disorders such as substance-related disorders,childhood disorders, dementia, autistic disorder, adjustment disorder,delirium, multi-infarct dementia, and Tourette's disorder as describedin Diagnostic and Statistical Manual of Mental Disorders, FourthEdition, (DSM IV). Typically, such disorders have a complex geneticand/or a biochemical component.

[0040] “A psychotic disorder” refers to a condition that affects themind, resulting in at least some loss of contact with reality. Symptomsof a psychotic disorder include, e.g., hallucinations, changed behaviorthat is not based on reality, delusions and the like. See, e.g., DSM IV.Schizophrenia, schizoaffective disorder, schizophreniform disorder,delusional disorder, brief psychotic disorder, substance-inducedpsychotic disorder, and shared psychotic disorder are examples ofpsychotic disorders.

[0041] “Schizophrenia” refers to a psychotic disorder involving awithdrawal from reality by an individual. Symptoms comprise for at leasta part of a month two or more of the following symptoms: delusions (onlyone symptom is required if a delusion is bizarre, such as being abductedin a space ship from the sun); hallucinations (only one symptom isrequired if hallucinations are of at least two voices talking to oneanother or of a voice that keeps up a running commentary on thepatient's thoughts or actions); disorganized speech (e.g., frequentderailment or incoherence); grossly disorganized or catatonic behavior;or negative symptoms, i.e., affective flattening, alogia, or avolition.Schizophrenia encompasses disorders such as, e.g., schizoaffectivedisorders. Diagnosis of schizophrenia is described in, e.g., DSM IV.Types of schizophrenia include, e.g., paranoid, disorganized, catatonic,undiffereentiated, and residual.

[0042] “mood disorder” refers to disruption of feeling tone or emotionalstate experienced by an individual for an extensive period of time. Mooddisorders include major depression disorder (i.e., unipolar disorder),mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and manyothers. See, e.g., Diagnostic and Statistical Manual of MentalDisorders, Fourth Edition, (DSM IV).

[0043] “Major depression disorder,” “major depressive disorder,” or“unipolar disorder” refers to a mood disorder involving any of thefollowing symptoms: persistent sad, anxious, or “empty” mood; feelingsof hopelessness or pessimism; feelings of guilt, worthlessness, orhelplessness; loss of interest or pleasure in hobbies and activitiesthat were once enjoyed, including sex; decreased energy, fatigue, being“slowed down”; difficulty concentrating, remembering, or makingdecisions; insomnia, early-morning awakening, or oversleeping; appetiteand/or weight loss or overeating and weight gain; thoughts of death orsuicide or suicide attempts; restlessness or irritability; or persistentphysical symptoms that do not respond to treatment, such as headaches,digestive disorders, and chronic pain. Various subtypes of depressionare described in, e.g., DSM IV.

[0044] “Bipolar disorder” is a mood disorder characterized byalternating periods of extreme moods. A person with bipolar disorderexperiences cycling of moods that usually swing from being overly elatedor irritable (mania) to sad and hopeless (depression) and then backagain, with periods of normal mood in between. Diagnosis of bipolardisorder is described in, e.g., DSM IV. Bipolar disorders includebipolar disorder I (mania with or without major depression) and bipolardisorder II (hypomania with major depression), see, e.g., DSM IV.

[0045] An “agonist” refers to an agent that binds to a polypeptide orpolynucleotide of the invention, stimulates, increases, activates,facilitates, enhances activation, sensitizes or up regulates theactivity or expression of a polypeptide or polynucleotide of theinvention.

[0046] An “antagonist” refers to an agent that inhibits expression of apolypeptide or polynucleotide of the invention or binds to, partially ortotally blocks stimulation, decreases, prevents, delays activation,inactivates, desensitizes, or down regulates the activity of apolypeptide or polynucleotide of the invention.

[0047] “Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity, e.g., ligands, agonists, antagonists, andtheir homologs and mimetics. The term “modulator” includes inhibitorsand activators. Inhibitors are agents that, e.g., inhibit expression ofa polypeptide or polynucleotide of the invention or bind to, partiallyor totally block stimulation or enzymatic activity, decrease, prevent,delay activation, inactivate, desensitize, or down regulate the activityof a polypeptide or polynucleotide of the invention, e.g., antagonists.Activators are agents that, e.g., induce or activate the expression of apolypeptide or polynucleotide of the invention or bind to, stimulate,increase, open, activate, facilitate, enhance activation or enzymaticactivity, sensitize or up regulate the activity of a polypeptide orpolynucleotide of the invention, e.g., agonists. Modulators includenaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Assays to identify inhibitors andactivators include, e.g., applying putative modulator compounds tocells, in the presence or absence of a polypeptide or polynucleotide ofthe invention and then determining the functional effects on apolypeptide or polynucleotide of the invention activity. Samples orassays comprising a polypeptide or polynucleotide of the invention thatare treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of effect. Control samples (untreatedwith modulators) are assigned a relative activity value of 100%.Inhibition is achieved when the activity value of a polypeptide orpolynucleotide of the invention relative to the control is about 80%,optionally 50% or 25-1%. Activation is achieved when the activity valueof a polypeptide or polynucleotide of the invention relative to thecontrol is 110%, optionally 150%, optionally 200-500%, or 1000-3000%higher.

[0048] The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid, fattyacid, polynucleotide, oligonucleotide, etc. The test compound can be inthe form of a library of test compounds, such as a combinatorial orrandomized library that provides a sufficient range of diversity. Testcompounds are optionally linked to a fusion partner, e.g., targetingcompounds, rescue compounds, dimerization compounds, stabilizingcompounds, addressable compounds, and other functional moieties.Conventionally, new chemical entities with useful properties aregenerated by identifying a test compound (called a “lead compound”) withsome desirable property or activity, e.g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis.

[0049] A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 Daltons and less than about 2500 Daltons, preferably lessthan about 2000 Daltons, preferably between about 100 to about 1000Daltons, more preferably between about 200 to about 500 Daltons.

[0050] “Determining the functional effect” refers to assaying for acompound that increases or decreases a parameter that is indirectly ordirectly under the influence of a polynucleotide or polypeptide of theinvention (such as a polynucleotide of Table 2, 3, or 4 or a polypeptideencoded by a gene of Table 2, 3, or 4), e.g., measuring physical andchemical or phenotypic effects. Such functional effects can be measuredby any means known to those skilled in the art, e.g., changes inspectroscopic (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility propertiesfor the protein; measuring inducible markers or transcriptionalactivation of the protein; measuring binding activity or binding assays,e.g. binding to antibodies; measuring changes in ligand bindingaffinity; measurement of calcium influx; measurement of the accumulationof an enzymatic product of a polypeptide of the invention or depletionof an substrate; measurement of changes in protein levels of apolypeptide of the invention; measurement of RNA stability; G-proteinbinding; GPCR phosphorylation or dephosphorylation; signal transduction,e.g., receptor-ligand interactions, second messenger concentrations(e.g., cAMP, IP3, or intracellular Ca²⁺); identification of downstreamor reporter gene expression (CAT, luciferase, β-gal, GFP and the like),e.g., via chemiluminescence, fluorescence, colorimetric reactions,antibody binding, inducible markers, and ligand binding assays.

[0051] Samples or assays comprising a nucleic acid or protein disclosedherein that are treated with a potential activator, inhibitor, ormodulator are compared to control samples without the inhibitor,activator, or modulator to examine the extent of inhibition. Controlsamples (untreated with inhibitors) are assigned a relative proteinactivity value of 100%. Inhibition is achieved when the activity valuerelative to the control is about 80%, preferably 50%, more preferably25-0%. Activation is achieved when the activity value relative to thecontrol (untreated with activators) is 110%, more preferably 150%, morepreferably 200-500% (i.e., two to five fold higher relative to thecontrol), more preferably 1000-3000% higher.

[0052] “Biological sample” includes sections of tissues such as biopsyand autopsy samples, and frozen sections taken for histologic purposes.Such samples include blood, spinal fluid, sputum, tissue, lysed cells,brain biopsy, cultured cells, e.g., primary cultures, explants, andtransformed cells, stool, urine, etc. A biological sample is typicallyobtained from a eukaryotic organism, most preferably a mammal such as aprimate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0053] “Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

[0054] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0055] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Paul (Ed.) Fundamental Immunology, ThirdEdition, Raven Press, NY (1993)). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

[0056] The terms “peptidomimetic” and “mimetic” refer to a syntheticchemical compound that has substantially the same structural andfunctional characteristics of the polynucleotides, polypeptides,antagonists or agonists of the invention. Peptide analogs are commonlyused in the pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of non-peptidecompound are termed “peptide mimetics” or “peptidomimetics” (Fauchere,Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporatedherein by reference). Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalent orenhanced therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity), such as a CCX CKR,but have one or more peptide linkages optionally replaced by a linkageselected from the group consisting of, e.g., —CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. The mimeticcan be either entirely composed of synthetic, non-natural analogues ofamino acids, or, is a chimeric molecule of partly natural peptide aminoacids and partly non-natural analogs of amino acids. The mimetic canalso incorporate any amount of natural amino acid conservativesubstitutions as long as such substitutions also do not substantiallyalter the mimetic's structure and/or activity. For example, a mimeticcomposition is within the scope of the invention if it is capable ofcarrying out the binding or enzymatic activities of a polypeptide orpolynucleotide of the invention or inhibiting or increasing theenzymatic activity or expression of a polypeptide or polynucleotide ofthe invention.

[0057] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0058] The term “isolated,” when applied to a nucleic acid or protein,denotes that the nucleic acid or protein is essentially free of othercellular components with which it is associated in the natural state. Itis preferably in a homogeneous state although it can be in either a dryor aqueous solution. Purity and homogeneity are typically determinedusing analytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

[0059] The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs (haplotypes), and complementary sequences as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)). The term nucleic acid is used interchangeably withgene, cDNA, and mRNA encoded by a gene.

[0060] The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

[0061] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0062] Amino acids may be referred to herein by either the commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0063] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

[0064] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0065] The following eight groups each contain amino acids that areconservative substitutions for one another:

[0066] 1) Alanine (A), Glycine (G);

[0067] 2) Aspartic acid (D), Glutamic acid (E);

[0068] 3) Asparagine (N), Glutamine (Q);

[0069] 4) Arginine (R), Lysine (K);

[0070] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0071] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0072] 7) Serine (S), Threonine (T); and

[0073] 8) Cysteine (C), Methionine (M)

[0074] (see, e.g., Creighton, Proteins (1984)).

[0075] “Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

[0076] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecomplement of a test sequence. Optionally, the identity exists over aregion that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesin length.

[0077] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0078] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Ausubel et al., Current Protocols in MolecularBiology (1995 supplement)).

[0079] An example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc.Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=4 and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and acomparison of both strands.

[0080] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0081] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

[0082] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0083] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes. Nucleic acids thathybridize to the genes listed in Tables 1-8 are encompassed by theinvention.

[0084] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15,30, 60, 120, or more minutes. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0085] For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C. depending on primer length. For highstringency PCR amplification, a temperature of about 62° C. is typical,although high stringency annealing temperatures can range from about 50°C. to about 65° C., depending on the primer length and specificity.Typical cycle conditions for both high and low stringency amplificationsinclude a denaturation phase of 90° C.-95° C. for 30 sec-2 min., anannealing phase lasting 30 sec.-2 min., and an extension phase of about72° C. for 1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications (1990).

[0086] The phrase “a nucleic acid sequence encoding” refers to a nucleicacid that contains sequence information for a structural RNA such asrRNA, a tRNA, or the primary amino acid sequence of a specific proteinor peptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences which may be introduced to conform with codon preference in aspecific host cell.

[0087] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all.

[0088] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0089] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0090] The phrase “specifically (or selectively) binds to an antibody”or “specifically (or selectively) immunoreactive with”, when referringto a protein or peptide, refers to a binding reaction which isdeterminative of the presence of the protein in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein and do not bind in a significant amount to otherproteins present in the sample. Specific binding to an antibody undersuch conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raisedagainst a protein having an amino acid sequence encoded by any of thepolynucleotides of the invention can be selected to obtain antibodiesspecifically immunoreactive with that protein and not with otherproteins, except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring HarborPublications, NY (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.Typically, a specific or selective reaction will be at least twice thebackground signal or noise and more typically more than 10 to 100 timesbackground.

[0091] One who is “predisposed for a mental disorder” as used hereinmeans a person who has an inclination or a higher likelihood ofdeveloping a mental disorder when compared to an average person in thegeneral population.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[0092] To understand the complex genetic basis of mental disorders, thepresent invention provides studies that have been conducted toinvestigate the expression patterns of genes that are differentiallyexpressed specifically in central nervous system of subjects with mooddisorders. The large spectrum of symptoms associated with mentaldisorders is likely a reflection of the complex genetic basis andcomplex gene expression patterns in patients with mental disorders.Different combinations of the genes disclosed herein can be responsiblefor one or more mental disorders. Furthermore, brain pathways orcircuits as well as subcellular pathways are important for understandingthe development and diagnosis of mental disorders. The selected brainregions described herein (AnCng, DLPFC, and CB) are implicated in theclinical symptoms of mental disorders such as mood disorders. Brainimaging studies focusing on particular brain regions, cytoarchitectualchanges in brain regions, expression of key neurotransmittors or relatedmolecules in brain regions, and subcellular pathways in brain regionsall contribute to the development of mental disorders, and thus are animportant consideration in the diagnosis and therapeutic uses describedherein.

[0093] The present invention demonstrates the altered expression (eitherhigher or lower expression) of the genes of Tables 1-8 at the mRNA levelin selected brain regions of patients diagnosed with mood disorders(e.g., bipolar disorder and major depression disorder) in comparisonwith normal individuals. This invention thus provides methods fordiagnosis of mental disorders such as mood disorders (e.g., bipolardisorder, major depression, and the like), psychotic disorders (e.g.,schizophrenia, and the like), and other mental disorders by detectingthe level of a transcript or translation product of the genes listed inTables 1-8 as well as their corresponding biochemical pathways. Thechromosomal location of such genes can be used to discover other genesin the region that are linked to development of a particular disorder.

[0094] The invention further provides methods of identifying a compounduseful for the treatment of such disorders by selecting compounds thatmodulates the functional effect of the translation products or theexpression of the transcripts described herein. The invention alsoprovides for methods of treating patients with such mental disorders,e.g., by administering the compounds of the invention or by genetherapy.

[0095] The genes and the polypeptides that they encode, which areassociated with mood disorders such as bipolar disease and majordepression, are useful for facilitating the design and development ofvarious molecular diagnostic tools such as GeneChips™ containing probesets specific for all or selected mental disorders, including but notlimited to mood disorders, and as an ante-and/or post-natal diagnostictool for screening newborns in concert with genetic counseling. Otherdiagnostic applications include evaluation of disease susceptibility,prognosis, and monitoring of disease or treatment process, as well asproviding individualized medicine via predictive drug profiling systems,e.g., by correlating specific genomic motifs with the clinical responseof a patient to individual drugs. In addition, the present invention isuseful for multiplex SNP or haplotype profiling, including but notlimited to the identification of pharmacogenetic targets at the gene,mRNA, protein, and pathway level.

[0096] The genes and the polypeptides that they encode, describedherein, as also useful as drug targets for the development oftherapeutic drugs for the treatment or prevention of mental disorders,including but not limited to mood disorders. Mental disorders have ahigh co-morbidity with other neurological disorders, such as Parkinson'sdisease or Alzheimer's. Therefore, the present invention can be used fordiagnosis and treatment of patients with multiple disease states thatinclude a mental disorder such as a mood disorder.

[0097] For example, antidepressants belong to different classes, e.g.,desipramine, bupropion, and fluoxetine are in general equally effect forthe treatment of clinical depression, but act by different mechanisms.The similar effectiveness of the drugs for treatment of mood disorderssuggests that they act through a yet as unidentified common pathway. Wedisclose herein that different classes of antidepressants (specificserotonin reuptake inhibitors, like fluoxetine and tricyclicantidepressants, like desipramine) regulate a common gene, and/or acommon group of genes as well as a unique set of genes when the humanand animal results herein are compared.

II. General Recombinant Nucleic Acid Methods for Use with the Invention

[0098] In numerous embodiments of the present invention, polynucleotidesof the invention will be isolated and cloned using recombinant methods.Such polynucleotides include, e.g., those listed in Tables 1-8, whichcan be used for, e.g., protein expression or during the generation ofvariants, derivatives, expression cassettes, to monitor gene expression,for the isolation or detection of sequences of the invention indifferent species, for diagnostic purposes in a patient, e.g., to detectmutations or to detect expression levels of nucleic acids orpolypeptides of the invention. In some embodiments, the sequences of theinvention are operably linked to a heterologous promoter. In oneembodiment, the nucleic acids of the invention are from any mammal,including, in particular, e.g., a human, a mouse, a rat, a primate, etc.

[0099] A. General Recombinant Nucleic Acids Methods

[0100] This invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

[0101] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

[0102] Oligonucleotides that are not commercially available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22:1859-1862 (1981), using an automated synthesizer, as describedin Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255:137-149 (1983).

[0103] The sequence of the cloned genes and synthetic oligonucleotidescan be verified after cloning using, e.g., the chain termination methodfor sequencing double-stranded templates of Wallace et al., Gene16:21-26 (1981).

[0104] B. Cloning Methods for the Isolation of Nucleotide SequencesEncoding Desired Proteins

[0105] In general, the nucleic acids encoding the subject proteins arecloned from DNA sequence libraries that are made to encode cDNA orgenomic DNA. The particular sequences can be located by hybridizing withan oligonucleotide probe, the sequence of which can be derived from thesequences of the genes listed in Tables 1-8, which provide a referencefor PCR primers and defines suitable regions for isolating specificprobes. Alternatively, where the sequence is cloned into an expressionlibrary, the expressed recombinant protein can be detectedimmunologically with antisera or purified antibodies made against apolypeptide comprising an amino acid sequence encoded by a gene listedin Table 1-8.

[0106] Methods for making and screening genomic and cDNA libraries arewell known to those of skill in the art (see, e.g., Gubler and HoffmanGene 25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977);and Sambrook, supra). Brain cells are an example of suitable cells toisolate RNA and cDNA sequences of the invention.

[0107] Briefly, to make the cDNA library, one should choose a sourcethat is rich in mRNA. The mRNA can then be made into cDNA, ligated intoa recombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. For a genomic library, the DNA isextracted from a suitable tissue and either mechanically sheared orenzymatically digested to yield fragments of preferably about 5-100 kb.The fragments are then separated by gradient centrifugation fromundesired sizes and are constructed in bacteriophage lambda vectors.These vectors and phage are packaged in vitro, and the recombinantphages are analyzed by plaque hybridization. Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

[0108] An alternative method combines the use of syntheticoligonucleotide primers with polymerase extension on an mRNA or DNAtemplate. Suitable primers can be designed from specific sequences ofthe invention. This polymerase chain reaction (PCR) method amplifies thenucleic acids encoding the protein of interest directly from mRNA, cDNA,genomic libraries or cDNA libraries. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acids encoding specific proteins and express said proteins, tosynthesize nucleic acids that will be used as probes for detecting thepresence of mRNA encoding a polypeptide of the invention inphysiological samples, for nucleic acid sequencing, or for otherpurposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes amplifiedby a PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

[0109] Appropriate primers and probes for identifying polynucleotides ofthe invention from mammalian tissues can be derived from the sequencesprovided herein. For a general overview of PCR, see, Innis et al. PCRProtocols: A Guide to Methods and Applications, Academic Press, SanDiego (1990).

[0110] Synthetic oligonucleotides can be used to construct genes. Thisis done using a series of overlapping oligonucleotides, usually 40-120bp in length, representing both the sense and anti-sense strands of thegene. These DNA fragments are then annealed, ligated and cloned.

[0111] A gene encoding a polypeptide of the invention can be clonedusing intermediate vectors before transformation into mammalian cellsfor expression. These intermediate vectors are typically prokaryotevectors or shuttle vectors. The proteins can be expressed in eitherprokaryotes, using standard methods well known to those of skill in theart, or eukaryotes as described infra.

III. Purification of Proteins of the Invention

[0112] Either naturally occurring or recombinant polypeptides of theinvention can be purified for use in functional assays. Naturallyoccurring polypeptides, e.g., polypeptides encoded by genes listed inTables 1-8, can be purified, for example, from mouse or human tissuesuch as brain or any other source of an ortholog. Recombinantpolypeptides can be purified from any suitable expression system.

[0113] The polypeptides of the invention may be purified to substantialpurity by standard techniques, including selective precipitation withsuch substances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

[0114] A number of procedures can be employed when recombinantpolypeptides are purified. For example, proteins having establishedmolecular adhesion properties can be reversible fused to polypeptides ofthe invention. With the appropriate ligand, the polypeptides can beselectively adsorbed to a purification column and then freed from thecolumn in a relatively pure form. The fused protein is then removed byenzymatic activity. Finally the polypeptide can be purified usingimmunoaffinity columns.

[0115] A. Purification of Proteins from Recombinant Bacteria

[0116] When recombinant proteins are expressed by the transformedbacteria in large amounts, typically after promoter induction, althoughexpression can be constitutive, the proteins may form insolubleaggregates. There are several protocols that are suitable forpurification of protein inclusion bodies. For example, purification ofaggregate proteins (hereinafter referred to as inclusion bodies)typically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells typically, but notlimited to, by incubation in a buffer of about 100-150 μg/ml lysozymeand 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can beground using a Polytron grinder (Brinkman Instruments, Westbury, N.Y.).Alternatively, the cells can be sonicated on ice. Alternate methods oflysing bacteria are described in Ausubel et al. and Sambrook et al.,both supra, and will be apparent to those of skill in the art.

[0117] The cell suspension is generally centrifuged and the pelletcontaining the inclusion bodies resuspended in buffer which does notdissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2),1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. Itmay be necessary to repeat the wash step to remove as much cellulardebris as possible. The remaining pellet of inclusion bodies may beresuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH6.8, 150 mM NaCl). Other appropriate buffers will be apparent to thoseof skill in the art.

[0118] Following the washing step, the inclusion bodies are solubilizedby the addition of a solvent that is both a strong hydrogen acceptor anda strong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for usein this procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of the immunologically and/or biologically activeprotein of interest. After solubilization, the protein can be separatedfrom other bacterial proteins by standard separation techniques.

[0119] Alternatively, it is possible to purify proteins from bacteriaperiplasm. Where the protein is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to those of skillin the art (see, Ausubel et al., supra). To isolate recombinant proteinsfrom the periplasm, the bacterial cells are centrifuged to form apellet. The pellet is resuspended in a buffer containing 20% sucrose. Tolyse the cells, the bacteria are centrifuged and the pellet isresuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath forapproximately 10 minutes. The cell suspension is centrifuged and thesupernatant decanted and saved. The recombinant proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

[0120] B. Standard Protein Separation Techniques For Purifying Proteins

[0121] 1. Solubility Fractionation

[0122] Often as an initial step, and if the protein mixture is complex,an initial salt fractionation can separate many of the unwanted hostcell proteins (or proteins derived from the cell culture media) from therecombinant protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol is to add saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This will precipitate the most hydrophobic proteins. Theprecipitate is discarded (unless the protein of interest is hydrophobic)and ammonium sulfate is added to the supernatant to a concentrationknown to precipitate the protein of interest. The precipitate is thensolubilized in buffer and the excess salt removed if necessary, througheither dialysis or diafiltration. Other methods that rely on solubilityof proteins, such as cold ethanol precipitation, are well known to thoseof skill in the art and can be used to fractionate complex proteinmixtures.

[0123] 2. Size Differential Filtration

[0124] Based on a calculated molecular weight, a protein of greater andlesser size can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

[0125] 3. Column Chromatography

[0126] The proteins of interest can also be separated from otherproteins on the basis of their size, net surface charge, hydrophobicityand affinity for ligands. In addition, antibodies raised againstproteins can be conjugated to column matrices and the proteinsimmunopurified. All of these methods are well known in the art.

[0127] It will be apparent to one of skill that chromatographictechniques can be performed at any scale and using equipment from manydifferent manufacturers (e.g., Pharmacia Biotech).

IV. Detection of Gene Expression

[0128] Those of skill in the art will recognize that detection ofexpression of polynucleotides of the invention has many uses. Forexample, as discussed herein, detection of the level of polypeptides orpolynucleotides of the invention in a patient is useful for diagnosingmental disorders including mood disorders or psychotic disorders or apredisposition for a mood disorder or psychotic disorder. Moreover,detection of gene expression is useful to identify modulators ofexpression of the polypeptides or polynucleotides of the invention.

[0129] A variety of methods of specific DNA and RNA measurement usingnucleic acid hybridization techniques are known to those of skill in theart (see, Sambrook, supra). Some methods involve an electrophoreticseparation (e.g., Southern blot for detecting DNA, and Northern blot fordetecting RNA), but measurement of DNA and RNA can also be carried outin the absence of electrophoretic separation (e.g., by dot blot).Southern blot of genomic DNA (e.g., from a human) can be used forscreening for restriction fragment length polymorphism (RFLP) to detectthe presence of a genetic disorder affecting a polypeptide of theinvention.

[0130] The selection of a nucleic acid hybridization format is notcritical. A variety of nucleic acid hybridization formats are known tothose skilled in the art. For example, common formats include sandwichassays and competition or displacement assays. Hybridization techniquesare generally described in Hames and Higgins Nucleic Acid Hybridization,A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl.Acad. Sci. U.S.A., 63:378-383 (1969); and John et al. Nature,223:582-587 (1969).

[0131] Detection of a hybridization complex may require the binding of asignal-generating complex to a duplex of target and probepolynucleotides or nucleic acids. Typically, such binding occurs throughligand and anti-ligand interactions as between a ligand-conjugated probeand an anti-ligand conjugated with a signal. The binding of the signalgeneration complex is also readily amenable to accelerations by exposureto ultrasonic energy.

[0132] The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor in some cases, by attachment to a radioactive label (see, e.g.,Tijssen, “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Burdon and vanKnippenberg Eds., Elsevier (1985), pp. 9-20).

[0133] The probes are typically labeled either directly, as withisotopes, chromophores, lumiphores, chromogens, or indirectly, such aswith biotin, to which a streptavidin complex may later bind. Thus, thedetectable labels used in the assays of the present invention can beprimary labels (where the label comprises an element that is detecteddirectly or that produces a directly detectable element) or secondarylabels (where the detected label binds to a primary label, e.g., as iscommon in immunological labeling). Typically, labeled signal nucleicacids are used to detect hybridization. Complementary nucleic acids orsignal nucleic acids may be labeled by any one of several methodstypically used to detect the presence of hybridized polynucleotides. Themost common method of detection is the use of autoradiography with ³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like.

[0134] Other labels include, e.g., ligands that bind to labeledantibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand. An introduction to labels, labeling procedures anddetection of labels is found in Polak and Van Noorden Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and inHaugland Handbook of Fluorescent Probes and Research Chemicals, acombined handbook and catalogue Published by Molecular Probes, Inc.(1996).

[0135] In general, a detector which monitors a particular probe or probecombination is used to detect the detection reagent label. Typicaldetectors include spectrophotometers, phototubes and photodiodes,microscopes, scintillation counters, cameras, film and the like, as wellas combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons of skillin the art. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

[0136] Most typically, the amount of RNA is measured by quantifying theamount of label fixed to the solid support by binding of the detectionreagent. Typically, the presence of a modulator during incubation willincrease or decrease the amount of label fixed to the solid supportrelative to a control incubation which does not comprise the modulator,or as compared to a baseline established for a particular reaction type.Means of detecting and quantifying labels are well known to those ofskill in the art.

[0137] In preferred embodiments, the target nucleic acid or the probe isimmobilized on a solid support. Solid supports suitable for use in theassays of the invention are known to those of skill in the art. As usedherein, a solid support is a matrix of material in a substantially fixedarrangement.

[0138] A variety of automated solid-phase assay techniques are alsoappropriate. For instance, very large scale immobilized polymer arrays(VLSIPS™), available from Affymetrix, Inc. (Santa Clara, Calif.) can beused to detect changes in expression levels of a plurality of genesinvolved in the same regulatory pathways simultaneously. See, Tijssen,supra., Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993)Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) NatureMedicine 2(7): 753-759.

[0139] Detection can be accomplished, for example, by using a labeleddetection moiety that binds specifically to duplex nucleic acids (e.g.,an antibody that is specific for RNA-DNA duplexes). One preferredexample uses an antibody that recognizes DNA-RNA heteroduplexes in whichthe antibody is linked to an enzyme (typically by recombinant orcovalent chemical bonding). The antibody is detected when the enzymereacts with its substrate, producing a detectable product. Coutlee etal. (1989) Analytical Biochemistry 181:153-162; Bogulavski (1986) et al.J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res.141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc.Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Immunol.19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidiet al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et al. (1989) J.Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, includinghomo and heteroduplexes. Kits comprising antibodies specific for DNA:RNAhybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville,Md.).

[0140] In addition to available antibodies, one of skill in the art caneasily make antibodies specific for nucleic acid duplexes using existingtechniques, or modify those antibodies that are commercially or publiclyavailable. In addition to the art referenced above, general methods forproducing polyclonal and monoclonal antibodies are known to those ofskill in the art (see, e.g., Paul (3rd ed.) Fundamental Immunology RavenPress, Ltd., NY (1993); Coligan Current Protocols in ImmunologyWiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory ManualCold Spring Harbor Press, NY (1988); Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y., (1986);and Kohler and Milstein Nature 256: 495-497 (1975)). Other suitabletechniques for antibody preparation include selection of libraries ofrecombinant antibodies in phage or similar vectors (see, Huse et al.Science 246:1275-1281 (1989); and Ward et al. Nature 341:544-546(1989)). Specific monoclonal and polyclonal antibodies and antisera willusually bind with a K_(D) of at least about 0.1 μM, preferably at leastabout 0.01 μM or better, and most typically and preferably, 0.001 μM orbetter.

[0141] The nucleic acids used in this invention can be either positiveor negative probes. Positive probes bind to their targets and thepresence of duplex formation is evidence of the presence of the target.Negative probes fail to bind to the suspect target and the absence ofduplex formation is evidence of the presence of the target. For example,the use of a wild type specific nucleic acid probe or PCR primers mayserve as a negative probe in an assay sample where only the nucleotidesequence of interest is present.

[0142] The sensitivity of the hybridization assays may be enhancedthrough use of a nucleic acid amplification system that multiplies thetarget nucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system, in particular RT-PCR or realtime PCR, and the ligase chain reaction (LCR) system. Other methodsrecently described in the art are the nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario) and Q BetaReplicase systems. These systems can be used to directly identifymutants where the PCR or LCR primers are designed to be extended orligated only when a selected sequence is present. Alternatively, theselected sequences can be generally amplified using, for example,nonspecific PCR primers and the amplified target region later probed fora specific sequence indicative of a mutation.

[0143] An alternative means for determining the level of expression ofthe nucleic acids of the present invention is in situ hybridization. Insitu hybridization assays are well known and are generally described inAngerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells or tissue, preferentially human cells ortissue from the cerebellum or the hippocampus, are fixed to a solidsupport, typically a glass slide. If DNA is to be probed, the cells aredenatured with heat or alkali. The cells are then contacted with ahybridization solution at a moderate temperature to permit annealing ofspecific probes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

V. Immunological Detection of the Polypeptides of the Invention

[0144] In addition to the detection of polynucleotide expression usingnucleic acid hybridization technology, one can also use immunoassays todetect polypeptides of the invention. Immunoassays can be used toqualitatively or quantitatively analyze polypeptides. A general overviewof the applicable technology can be found in Harlow & Lane, Antibodies:A Laboratory Manual (1988).

[0145] A. Antibodies to Target Polypeptides or Other Immunogens

[0146] Methods for producing polyclonal and monoclonal antibodies thatreact specifically with a protein of interest or other immunogen areknown to those of skill in the art (see, e.g., Coligan, supra; andHarlow and Lane, supra; Stites et al., supra and references citedtherein; Goding, supra; and Kohler and Milstein Nature, 256:495-497(1975)). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors (see, Huse et al., supra; and Ward et al., supra). For example,in order to produce antisera for use in an immunoassay, the protein ofinterest or an antigenic fragment thereof, is isolated as describedherein. For example, a recombinant protein is produced in a transformedcell line. An inbred strain of mice or rabbits is immunized with theprotein using a standard adjuvant, such as Freund's adjuvant, and astandard immunization protocol. Alternatively, a synthetic peptidederived from the sequences disclosed herein and conjugated to a carrierprotein can be used as an immunogen.

[0147] Polyclonal sera are collected and titered against the immunogenin an immunoassay, for example, a solid phase immunoassay with theimmunogen immobilized on a solid support. Polyclonal antisera with atiter of 10⁴ or greater are selected and tested for theircross-reactivity against unrelated proteins or even other homologousproteins from other organisms, using a competitive binding immunoassay.Specific monoclonal and polyclonal antibodies and antisera will usuallybind with a K_(D) of at least about 0.1 mM, more usually at least about1 μM, preferably at least about 0.1 μM or better, and most preferably,0.01 μM or better.

[0148] A number of proteins of the invention comprising immunogens maybe used to produce antibodies specifically or selectively reactive withthe proteins of interest. Recombinant protein is the preferred immunogenfor the production of monoclonal or polyclonal antibodies. Naturallyoccurring protein, such as one comprising an amino acid sequence encodedby a gene listed in Table 1-8 may also be used either in pure or impureform. Synthetic peptides made using the protein sequences describedherein may also be used as an immunogen for the production of antibodiesto the protein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells and purified as generally described supra. The productis then injected into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated for subsequent usein immunoassays to measure the protein.

[0149] Methods of production of polyclonal antibodies are known to thoseof skill in the art. In brief, an immunogen, preferably a purifiedprotein, is mixed with an adjuvant and animals are immunized. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to thepolypeptide of interest. When appropriately high titers of antibody tothe immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein can be done if desired (see,Harlow and Lane, supra).

[0150] Monoclonal antibodies may be obtained using various techniquesfamiliar to those of skill in the art. Typically, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., supra.

[0151] Once target protein specific antibodies are available, theprotein can be measured by a variety of immunoassay methods withqualitative and quantitative results available to the clinician. For areview of immunological and immunoassay procedures in general see,Stites, supra. Moreover, the immunoassays of the present invention canbe performed in any of several configurations, which are reviewedextensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Fla.(1980); Tijssen, supra; and Harlow and Lane, supra.

[0152] Immunoassays to measure target proteins in a human sample may usea polyclonal antiserum that was raised to the protein (e.g., one has anamino acid sequence encoded by a gene listed in Table 1-8) or a fragmentthereof. This antiserum is selected to have low cross-reactivity againstdifferent proteins and any such cross-reactivity is removed byimmunoabsorption prior to use in the immunoassay.

[0153] B. Immunological Binding Assays

[0154] In a preferred embodiment, a protein of interest is detectedand/or quantified using any of a number of well-known immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Asai Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Academic Press, Inc. NY (1993); Stites, supra. Immunological bindingassays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte (in this case apolypeptide of the present invention or antigenic subsequences thereof).The capture agent is a moiety that specifically binds to the analyte. Ina preferred embodiment, the capture agent is an antibody thatspecifically binds, for example, a polypeptide of the invention. Theantibody may be produced by any of a number of means well known to thoseof skill in the art and as described above.

[0155] Immunoassays also often utilize a labeling agent to specificallybind to and label the binding complex formed by the capture agent andthe analyte. The labeling agent may itself be one of the moietiescomprising the antibody/analyte complex. Alternatively, the labelingagent may be a third moiety, such as another antibody, that specificallybinds to the antibody/protein complex.

[0156] In a preferred embodiment, the labeling agent is a secondantibody bearing a label. Alternatively, the second antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second antibody can be modified with a detectable moiety,such as biotin, to which a third labeled molecule can specifically bind,such as enzyme-labeled streptavidin.

[0157] Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G, can also be used asthe label agents. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non-immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see, generally, Kronval, et al. J. Immunol., 111:1401-1406(1973); and Akerstrom, et al. J. Immunol., 135:2589-2542 (1985)).

[0158] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. The incubation time will depend upon the assayformat, analyte, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0159] 1. Non-Competitive Assay Formats

[0160] Immunoassays for detecting proteins of interest from tissuesamples may be either competitive or noncompetitive. Noncompetitiveimmunoassays are assays in which the amount of captured analyte (in thiscase the protein) is directly measured. In one preferred “sandwich”assay, for example, the capture agent (e.g., antibodies specific for apolypeptide encoded by a gene listed in Table 1-8) can be bound directlyto a solid substrate where it is immobilized. These immobilizedantibodies then capture the polypeptide present in the test sample. Thepolypeptide thus immobilized is then bound by a labeling agent, such asa second antibody bearing a label. Alternatively, the second antibodymay lack a label, but it may, in turn, be bound by a labeled thirdantibody specific to antibodies of the species from which the secondantibody is derived. The second can be modified with a detectablemoiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

[0161] 2. Competitive Assay Formats

[0162] In competitive assays, the amount of analyte (such as apolypeptide encoded by a gene listed in Table 1-8) present in the sampleis measured indirectly by measuring the amount of an added (exogenous)analyte displaced (or competed away) from a capture agent (e.g., anantibody specific for the analyte) by the analyte present in the sample.In one competitive assay, a known amount of, in this case, the proteinof interest is added to the sample and the sample is then contacted witha capture agent, in this case an antibody that specifically binds to apolypeptide of the invention: The amount of immunogen bound to theantibody is inversely proportional to the concentration of immunogenpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. For example, the amount ofthe polypeptide bound to the antibody may be determined either bymeasuring the amount of subject protein present in a protein/antibodycomplex or, alternatively, by measuring the amount of remaininguncomplexed protein. The amount of protein may be detected by providinga labeled protein molecule.

[0163] Immunoassays in the competitive binding format can be used forcross-reactivity determinations. For example, a protein of interest canbe immobilized on a solid support. Proteins are added to the assay whichcompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to that of the proteinof interest. The percent cross-reactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% cross-reactivity with each of the proteins listed above are selectedand pooled. The cross-reacting antibodies are optionally removed fromthe pooled antisera by immunoabsorption with the considered proteins,e.g., distantly related homologs.

[0164] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein, thought to be perhaps a protein of the present invention, tothe immunogen protein. In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required is less than 10 times the amount of the proteinpartially encoded by a sequence herein that is required, then the secondprotein is said to specifically bind to an antibody generated to animmunogen consisting of the target protein.

[0165] 3. Other Assay Formats

[0166] In a particularly preferred embodiment, western blot (immunoblot)analysis is used to detect and quantify the presence of a polypeptide ofthe invention in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support (such as, e.g., a nitrocellulose filter, a nylon filter,or a derivatized nylon filter) and incubating the sample with theantibodies that specifically bind the protein of interest. For example,the antibodies specifically bind to a polypeptide of interest on thesolid support. These antibodies may be directly labeled or alternativelymay be subsequently detected using labeled antibodies (e.g., labeledsheep anti-mouse antibodies) that specifically bind to the antibodiesagainst the protein of interest.

[0167] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

[0168] 4. Labels

[0169] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most labels useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

[0170] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on the sensitivity required, the ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

[0171] Non-radioactive labels are often attached by indirect means. Themolecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorescent compound.A variety of enzymes and fluorescent compounds can be used with themethods of the present invention and are well-known to those of skill inthe art (for a review of various labeling or signal producing systemswhich may be used, see, e.g., U.S. Pat. No. 4,391,904).

[0172] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge-coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected directly by observing the color associated withthe label. Thus, in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

[0173] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need to be labeled and the presence ofthe target antibody is detected by simple visual inspection.

VI. Screening for Modulators of Polypeptides and Polynucleotides of theInvention

[0174] Modulators of polypeptides or polynucleotides of the invention,i.e. agonists or antagonists of their activity or modulators ofpolypeptide or polynucleotide expression, are useful for treating anumber of human diseases, including mood disorders or psychoticdisorders. Administration of agonists, antagonists or other agents thatmodulate expression of the polynucleotides or polypeptides of theinvention can be used to treat patients with mood disorders or psychoticdisorders.

[0175] A. Screening Methods

[0176] A number of different screening protocols can be utilized toidentify agents that modulate the level of expression or activity ofpolypeptides and polynucleotides of the invention in cells, particularlymammalian cells, and especially human cells. In general terms, thescreening methods involve screening a plurality of agents to identify anagent that modulates the polypeptide activity by binding to apolypeptide of the invention, modulating inhibitor binding to thepolypeptide or activating expression of the polypeptide orpolynucleotide, for example.

[0177] 1. Binding Assays

[0178] Preliminary screens can be conducted by screening for agentscapable of binding to a polypeptide of the invention, as at least someof the agents so identified are likely modulators of polypeptideactivity. The binding assays usually involve contacting a polypeptide ofthe invention with one or more test agents and allowing sufficient timefor the protein and test agents to form a binding complex. Any bindingcomplexes formed can be detected using any of a number of establishedanalytical techniques. Protein binding assays include, but are notlimited to, methods that measure co-precipitation, co-migration onnon-denaturing SDS-polyacrylamide gels, and co-migration on Westernblots (see, e.g., Bennet and Yamamura, (1985) “Neurotransmitter, Hormoneor Drug Receptor Binding Methods,” in Neurotransmitter Receptor Binding(Yamamura, H. I., et al., eds.), pp. 61-89. The protein utilized in suchassays can be naturally expressed, cloned or synthesized.

[0179] Binding assays are also useful, e.g., for identifying endogenousproteins that interact with a polypeptide of the invention. For example,antibodies, receptors or other molecules that bind a polypeptide of theinvention can be identified in binding assays.

[0180] 2. Expression Assays

[0181] Certain screening methods involve screening for a compound thatup or down-regulates the expression of a polypeptide or polynucleotideof the invention. Such methods generally involve conducting cell-basedassays in which test compounds are contacted with one or more cellsexpressing a polypeptide or polynucleotide of the invention and thendetecting an increase or decrease in expression (either transcript,translation product, or catalytic product). Some assays are performedwith peripheral cells, or other cells, that express an endogenouspolypeptide or polynucleotide of the invention.

[0182] Polypeptide or polynucleotide expression can be detected in anumber of different ways. As described infra, the expression level of apolynucleotide of the invention in a cell can be determined by probingthe mRNA expressed in a cell with a probe that specifically hybridizeswith a transcript (or complementary nucleic acid derived therefrom) of apolynucleotide of the invention. Probing can be conducted by lysing thecells and conducting Northern blots or without lysing the cells using insitu-hybridization techniques. Alternatively, a polypeptide of theinvention can be detected using immunological methods in which a celllysate is probed with antibodies that specifically bind to a polypeptideof the invention.

[0183] Other cell-based assays are reporter assays conducted with cellsthat do not express a polypeptide or polynucleotide of the invention.Certain of these assays are conducted with a heterologous nucleic acidconstruct that includes a promoter of a polynucleotide of the inventionthat is operably linked to a reporter gene that encodes a detectableproduct. A number of different reporter genes can be utilized. Somereporters are inherently detectable. An example of such a reporter isgreen fluorescent protein that emits fluorescence that can be detectedwith a fluorescence detector. Other reporters generate a detectableproduct. Often such reporters are enzymes. Exemplary enzyme reportersinclude, but are not limited to, β-glucuronidase, chloramphenicol acetyltransferase (CAT); Alton and Vapnek (1979) Nature 282:864-869),luciferase, β-galactosidase, green fluorescent protein (GFP) andalkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238;and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).

[0184] In these assays, cells harboring the reporter construct arecontacted with a test compound. A test compound that either activatesthe promoter by binding to it or triggers a cascade that produces amolecule that activates the promoter causes expression of the detectablereporter. Certain other reporter assays are conducted with cells thatharbor a heterologous construct that includes a transcriptional controlelement that activates expression of a polynucleotide of the inventionand a reporter operably linked thereto. Here, too, an agent that bindsto the transcriptional control element to activate expression of thereporter or that triggers the formation of an agent that binds to thetranscriptional control element to activate reporter expression, can beidentified by the generation of signal associated with reporterexpression.

[0185] The level of expression or activity can be compared to a baselinevalue. As indicated above, the baseline value can be a value for acontrol sample or a statistical value that is representative ofexpression levels for a control population (e.g., healthy individualsnot having or at risk for mood disorders or psychotic disorders).Expression levels can also be determined for cells that do not express apolynucleotide of the invention as a negative control. Such cellsgenerally are otherwise substantially genetically the same as the testcells.

[0186] A variety of different types of cells can be utilized in thereporter assays. Cells that express an endogenous polypeptide orpolynucleotide of the invention include, e.g., brain cells, includingcells from the cerebellum, anterior cingulate cortex, or dorsolateralprefrontal cortex. Such brain regions are part of brain circuits orpathways that are implicated in mood disorders. Cells that do notendogenously express polynucleotides of the invention can beprokaryotic, but are preferably eukaryotic. The eukaryotic cells can beany of the cells typically utilized in generating cells that harborrecombinant nucleic acid constructs. Exemplary eukaryotic cells include,but are not limited to, yeast, and various higher eukaryotic cells suchas the COS, CHO and HeLa cell lines, and stem cells.

[0187] Various controls can be conducted to ensure that an observedactivity is authentic including running parallel reactions with cellsthat lack the reporter construct or by not contacting a cell harboringthe reporter construct with test compound. Compounds can also be furthervalidated as described below.

[0188] 3. Catalytic Activity

[0189] Catalytic activity of polypeptides of the invention can bedetermined by measuring the production of enzymatic products or bymeasuring the consumption of substrates. Activity refers to either therate of catalysis or the ability to the polypeptide to bind (K_(m)) thesubstrate or release the catalytic product (K_(d)).

[0190] Analysis of the activity of polypeptides of the invention areperformed according to general biochemical analyses. Such assays includecell-based assays as well as in vitro assays involving purified orpartially purified polypeptides or crude cell lysates. The assaysgenerally involve providing a known quantity of substrate andquantifying product as a function of time.

[0191] 4. Validation

[0192] Agents that are initially identified by any of the foregoingscreening methods can be further tested to validate the apparentactivity. Preferably such studies are conducted with suitable animalmodels. The basic format of such methods involves administering a leadcompound identified during an initial screen to an animal that serves asa model for humans and then determining if expression or activity of apolynucleotide or polypeptide of the invention is in fact upregulated.The animal models utilized in validation studies generally are mammalsof any kind. Specific examples of suitable animals include, but are notlimited to, primates, mice, and rats.

[0193] 5. Animal Models

[0194] Animal models of mental disorders also find use in screening formodulators. In one embodiment, rat models of depression (both chronicand acute), in which the rats are subjected to stress, are used forscreening. In one embodiment, invertebrate models such as Drosophilamodels can be used, screening for modulators of Drosophila orthologs ofthe human genes disclosed herein. In another embodiment, transgenicanimal technology including gene knockout technology, for example as aresult of homologous recombination with an appropriate gene targetingvector, or gene overexpression, will result in the absence, decreased orincreased expression of a polynucleotide or polypeptide of theinvention. The same technology can also be applied to make knockoutcells. When desired, tissue-specific expression or knockout of apolynucleotide or polypeptide of the invention may be necessary.Transgenic animals generated by such methods find use as animal modelsof mental disorders and are useful in screening for modulators of mentaldisorders.

[0195] Knockout cells and transgenic mice can be made by insertion of amarker gene or other heterologous gene into an endogenous gene site inthe mouse genome via homologous recombination. Such mice can also bemade by substituting an endogenous polynucleotide of the invention witha mutated version of the polynucleotide, or by mutating an endogenouspolynucleotide, e.g., by exposure to carcinogens.

[0196] For development of appropriate stem cells, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells partially derivedfrom the mutant cell line. Therefore, by breeding the chimeric mice itis possible to obtain a new line of mice containing the introducedgenetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)).Chimeric targeted mice can be derived according to Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).

[0197] B. Modulators of Polypeptides or Polynucleotides of the Invention

[0198] The agents tested as modulators of the polypeptides orpolynucleotides of the invention can be any small chemical compound, ora biological entity, such as a protein, sugar, nucleic acid or lipid.Alternatively, modulators can be genetically altered versions of apolypeptide or polynucleotide of the invention. Typically, testcompounds will be small chemical molecules and peptides. Essentially anychemical compound can be used as a potential modulator or ligand in theassays of the invention, although most often compounds that can bedissolved in aqueous or organic (especially DMSO-based) solutions areused. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland) and the like. Modulators also include agentsdesigned to reduce the level of mRNA of the invention (e.g. antisensemolecules, ribozymes, DNAzymes and the like) or the level of translationfrom an mRNA.

[0199] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0200] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0201] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. No. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

[0202] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A AppliedBiosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Tripos,Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.; MartekBiosciences, Columbia, Md., etc.).

[0203] C. Solid State and Soluble High Throughput Assays

[0204] In the high throughput assays of the invention, it is possible toscreen up to several thousand different modulators or ligands in asingle day. In particular, each well of a microtiter plate can be usedto run a separate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsare possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped.

[0205] The molecule of interest can be bound to the solid statecomponent, directly or indirectly, via covalent or non-covalent linkage,e.g., via a tag. The tag can be any of a variety of components. Ingeneral, a molecule that binds the tag (a tag binder) is fixed to asolid support, and the tagged molecule of interest is attached to thesolid support by interaction of the tag and the tag binder.

[0206] A number of tags and tag binders can be used, based upon knownmolecular interactions well described in the literature. For example,where a tag has a natural binder, for example, biotin, protein A, orprotein G, it can be used in conjunction with appropriate tag binders(avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin,etc.). Antibodies to molecules with natural binders such as biotin arealso widely available and appropriate tag binders (see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

[0207] Similarly, any haptenic or antigenic compound can be used incombination with an appropriate antibody to form a tag/tag binder pair.Thousands of specific antibodies are commercially available and manyadditional antibodies are described in the literature. For example, inone common configuration, the tag is a first antibody and the tag binderis a second antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs, such as agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

[0208] Synthetic polymers, such as polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, and polyacetates can also form anappropriate tag or tag binder. Many other tag/tag binder pairs are alsouseful in assay systems described herein, as would be apparent to one ofskill upon review of this disclosure.

[0209] Common linkers such as peptides, polyethers, and the like canalso serve as tags, and include polypeptide sequences, such as poly-Glysequences of between about 5 and 200 amino acids. Such flexible linkersare known to those of skill in the art. For example, poly(ethelyneglycol) linkers are available from Shearwater Polymers, Inc.,Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterofunctional linkages.

[0210] Tag binders are fixed to solid substrates using any of a varietyof methods currently available. Solid substrates are commonlyderivatized or functionalized by exposing all or a portion of thesubstrate to a chemical reagent which fixes a chemical group to thesurface which is reactive with a portion of the tag binder. For example,groups which are suitable for attachment to a longer chain portion wouldinclude amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanesand hydroxyalkylsilanes can be used to functionalize a variety ofsurfaces, such as glass surfaces. The construction of such solid phasebiopolymer arrays is well described in the literature (see, e.g.,Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solidphase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth.102:259-274 (1987) (describing synthesis of solid phase components onpins); Frank and Doring, Tetrahedron 44:60316040 (1988) (describingsynthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759(1996) (all describing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

[0211] The invention provides in vitro assays for identifying, in a highthroughput format, compounds that can modulate the expression oractivity of the polynucleotides or polypeptides of the invention. In apreferred embodiment, the methods of the invention include such acontrol reaction. For each of the assay formats described, “nomodulator” control reactions that do not include a modulator provide abackground level of binding activity.

[0212] In some assays it will be desirable to have positive controls toensure that the components of the assays are working properly. At leasttwo types of positive controls are appropriate. First, a known activatorof a polynucleotide or polypeptide of the invention can be incubatedwith one sample of the assay, and the resulting increase in signalresulting from an increased expression level or activity ofpolynucleotide or polypeptide determined according to the methodsherein. Second, a known inhibitor of a polynucleotide or polypeptide ofthe invention can be added, and the resulting decrease in signal for theexpression or activity can be similarly detected.

[0213] D. Computer-Based Assays

[0214] Yet another assay for compounds that modulate the activity of apolypeptide or polynucleotide of the invention involves computerassisted drug design, in which a computer system is used to generate athree-dimensional structure of the polypeptide or polynucleotide basedon the structural information encoded by its amino acid or nucleotidesequence. The input sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the molecule. Similaranalyses can be performed on potential receptors or binding partners ofthe polypeptides or polynucleotides of the invention. The models of theprotein or nucleotide structure are then examined to identify regions ofthe structure that have the ability to bind, e.g., a polypeptide orpolynucleotide of the invention. These regions are then used to identifypolypeptides that bind to a polypeptide or polynucleotide of theinvention.

[0215] The three-dimensional structural model of a protein is generatedby entering protein amino acid sequences of at least 10 amino acidresidues or corresponding nucleic acid sequences encoding a potentialreceptor into the computer system. The amino acid sequences encoded bythe nucleic acid sequences provided herein represent the primarysequences or subsequences of the proteins, which encode the structuralinformation of the proteins. At least 10 residues of an amino acidsequence (or a nucleotide sequence encoding 10 amino acids) are enteredinto the computer system from computer keyboards, computer readablesubstrates that include, but are not limited to, electronic storagemedia (e.g., magnetic diskettes, tapes, cartridges, and chips), opticalmedia (e.g., CD ROM), information distributed by internet sites, and byRAM. The three-dimensional structural model of the protein is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art.

[0216] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary, andquaternary structure of the protein of interest. The software looks atcertain parameters encoded by the primary sequence to generate thestructural model. These parameters are referred to as “energy terms,”and primarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

[0217] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0218] Once the structure has been generated, potential ligand bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of a polypeptide or polynucleotide of the invention toidentify binding sites of the polypeptide or polynucleotide of theinvention. Binding affinity between the protein and ligands isdetermined using energy terms to determine which ligands have anenhanced probability of binding to the protein.

[0219] Computer systems are also used to screen for mutations,polymorphic variants, alleles and interspecies homologs of genesencoding a polypeptide or polynucleotide of the invention. Suchmutations can be associated with disease states or genetic traits andcan be used for diagnosis. As described above, GeneChip™ and relatedtechnology can also be used to screen for mutations, polymorphicvariants, alleles and interspecies homologs. Once the variants areidentified, diagnostic assays can be used to identify patients havingsuch mutated genes. Identification of the mutated a polypeptide orpolynucleotide of the invention involves receiving input of a firstamino acid sequence of a polypeptide of the invention (or of a firstnucleic acid sequence encoding a polypeptide of the invention), e.g.,any amino acid sequence having at least 60%, optionally at least 70% or85%, identity with the amino acid sequence of interest, orconservatively modified versions thereof. The sequence is entered intothe computer system as described above. The first nucleic acid or aminoacid sequence is then compared to a second nucleic acid or amino acidsequence that has substantial identity to the first sequence. The secondsequence is entered into the computer system in the manner describedabove. Once the first and second sequences are compared, nucleotide oramino acid differences between the sequences are identified. Suchsequences can represent allelic differences in various polynucleotides,including SNPs and/or haplotypes, of the invention, and mutationsassociated with disease states and genetic traits.

VII. Compositions, Kits and Integrated Systems

[0220] The invention provides compositions, kits and integrated systemsfor practicing the assays described herein using polypeptides orpolynucleotides of the invention, antibodies specific for polypeptidesor polynucleotides of the invention, etc.

[0221] The invention provides assay compositions for use in solid phaseassays; such compositions can include, for example, one or morepolynucleotides or polypeptides of the invention immobilized on a solidsupport, and a labeling reagent. In each case, the assay compositionscan also include additional reagents that are desirable forhybridization. Modulators of expression or activity of polynucleotidesor polypeptides of the invention can also be included in the assaycompositions.

[0222] The invention also provides kits for carrying out the therapeuticand diagnostic assays of the invention. The kits typically include aprobe that comprises an antibody that specifically binds to polypeptidesor polynucleotides of the invention, and a label for detecting thepresence of the probe. The kits may include several polynucleotidesequences encoding polypeptides of the invention. Kits can include anyof the compositions noted above, and optionally further includeadditional components such as instructions to practice a high-throughputmethod of assaying for an effect on expression of the genes encoding thepolypeptides of the invention, or on activity of the polypeptides of theinvention, one or more containers or compartments (e.g., to hold theprobe, labels, or the like), a control modulator of the expression oractivity of polypeptides of the invention, a robotic armature for mixingkit components or the like.

[0223] The invention also provides integrated systems forhigh-throughput screening of potential modulators for an effect on theexpression or activity of the polypeptides of the invention. The systemstypically include a robotic armature which transfers fluid from a sourceto a destination, a controller which controls the robotic armature, alabel detector, a data storage unit which records label detection, andan assay component such as a microtiter dish comprising a well having areaction mixture or a substrate comprising a fixed nucleic acid orimmobilization moiety.

[0224] A number of robotic fluid transfer systems are available, or caneasily be made from existing components. For example, a Zymate XP(Zymark Corporation; Hopkinton, Mass.) automated robot using a Microlab2200 (Hamilton; Reno, Nev.) pipetting station can be used to transferparallel samples to 96 well microtiter plates to set up several parallelsimultaneous STAT binding assays.

[0225] Optical images viewed (and, optionally, recorded) by a camera orother recording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image, e.g., using PC, MACINTOSH®, or UNIX® based(e.g., SUN® work station) computers.

[0226] One conventional system carries light from the specimen field toa cooled charge-coupled device (CCD) camera, in common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques. Lasar based systems can also be used.

VIII. Administration and Pharmaceutical Compositions

[0227] Modulators of the polynucleotides or polypeptides of theinvention (e.g., antagonists or agonists) can be administered directlyto a mammalian subject for modulation of activity of those molecules invivo. Administration is by any of the routes normally used forintroducing a modulator compound into ultimate contact with the tissueto be treated and is well known to those of skill in the art. Althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

[0228] Diseases that can be treated include the following, which includethe corresponding reference number from Morrison, DSM-IV Made Easy,1995: Schizophrenia, Catatonic, Subchronic, (295.21); Schizophrenia,Catatonic, Chronic (295.22); Schizophrenia, Catatonic, Subchronic withAcute Exacerbation (295.23); Schizophrenia, Catatonic, Chronic withAcute Exacerbation (295.24); Schizophrenia, Catatonic, in Remission(295.55); Schizophrenia, Catatonic, Unspecified (295.20); Schizophrenia,Disorganized, Subchronic (295.11); Schizophrenia, Disorganized, Chronic(295.12); Schizophrenia, Disorganized, Subchronic with AcuteExacerbation (295.13); Schizophrenia, Disorganized, Chronic with AcuteExacerbation (295.14); Schizophrenia, Disorganized, in Remission(295.15); Schizophrenia, Disorganized, Unspecified (295.10);Schizophrenia, Paranoid, Subchronic (295.31); Schizophrenia, Paranoid,Chronic (295.32); Schizophrenia, Paranoid, Subchronic with AcuteExacerbation (295.33); Schizophrenia, Paranoid, Chronic with AcuteExacerbation (295.34); Schizophrenia, Paranoid, in Remission (295.35);Schizophrenia, Paranoid, Unspecified (295.30); Schizophrenia,Undifferentiated, Subchronic (295.91); Schizophrenia, Undifferentiated,Chronic (295.92); Schizophrenia, Undifferentiated, Subchronic with AcuteExacerbation (295.93); Schizophrenia, Undifferentiated, Chronic withAcute Exacerbation (295.94); Schizophrenia, Undifferentiated, inRemission (295.95); Schizophrenia, Undifferentiated, Unspecified(295.90); Schizophrenia, Residual, Subchronic (295.61); Schizophrenia,Residual, Chronic (295.62); Schizophrenia, Residual, Subchronic withAcute Exacerbation (295.63); Schizophrenia, Residual, Chronic with AcuteExacerbation (295.94); Schizophrenia, Residual, in Remission (295.65);Schizophrenia, Residual, Unspecified (295.60); Delusional (Paranoid)Disorder (297.10); Brief Reactive Psychosis (298.80); SchizophreniformDisorder (295.40); Schizoaffective Disorder (295.70); Induced PsychoticDisorder (297.30); Psychotic Disorder NOS (Atypical Psychosis) (298.90);Personality Disorders, Paranoid (301.00); Personality Disorders,Schizoid (301.20); Personality Disorders, Schizotypal (301.22);Personality Disorders, Antisocial (301.70); Personality Disorders,Borderline (301.83) and bipolar disorders, maniac, hypomaniac, dysthymicor cyclothymic disorders, substance-induced mood disorders, majordepression, psychotic disorders, including paranoid psychosis, catatonicpsychosis, delusional psychosis, having schizoaffective disorder, andsubstance-induced psychotic disorder.

[0229] In some embodiments, modulators of polynucleotides orpolypeptides of the invention can be combined with other drugs usefulfor treating mental disorders including useful for treating mooddisorders, e.g., schizophrenia, bipolar disorders, or major depression.In some preferred embodiments, pharmaceutical compositions of theinvention comprise a modulator of a polypeptide of polynucleotide of theinvention combined with at least one of the compounds useful fortreating schizophrenia, bipolar disorder, or major depression, e.g.,such as those described in U.S. Pat. Nos. 6,297,262; 6,284,760;6,284,771; 6,232,326; 6,187,752; 6,117,890; 6,239,162 or 6,166,008.

[0230] The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

[0231] The modulators (e.g., agonists or antagonists) of the expressionor activity of the a polypeptide or polynucleotide of the invention,alone or in combination with other suitable components, can be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation or in compositions useful for injection. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

[0232] Formulations suitable for administration include aqueous andnon-aqueous solutions, isotonic sterile solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic, and aqueous and non-aqueous sterile suspensionsthat can include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, orally, nasally,topically, intravenously, intraperitoneally, or intrathecally. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. Solutions and suspensionscan be prepared from sterile powders, granules, and tablets of the kindpreviously described. The modulators can also be administered as part ofa prepared food or drug.

[0233] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the mental disorder. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side effects thataccompany the administration of a particular compound or vector in aparticular subject.

[0234] In determining the effective amount of the modulator to beadministered a physician may evaluate circulating plasma levels of themodulator, modulator toxicity, and the production of anti-modulatorantibodies. In general, the dose equivalent of a modulator is from about1 ng/kg to 10 mg/kg for a typical subject.

[0235] For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside effects of the modulator at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

IX. Gene Therapy Applications

[0236] A variety of human diseases can be treated by therapeuticapproaches that involve stably introducing a gene into a human cell suchthat the gene is transcribed and the gene product is produced in thecell. Diseases amenable to treatment by this approach include inheriteddiseases, including those in which the defect is in a single or multiplegenes. Gene therapy is also useful for treatment of acquired diseasesand other conditions. For discussions on the application of gene therapytowards the treatment of genetic as well as acquired diseases, see,Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932(1993).

[0237] In the context of the present invention, gene therapy can be usedfor treating a variety of disorders and/or diseases in which thepolynucleotides and polypeptides of the invention has been implicated.For example, compounds, including polynucleotides, can be identified bythe methods of the present invention as effective in treating a mentaldisorder. Introduction by gene therapy of these polynucleotides can thenbe used to treat, e.g., mental disorders including mood disorders andpsychotic disorders.

[0238] A. Vectors for Gene Delivery

[0239] For delivery to a cell or organism, the polynucleotides of theinvention can be incorporated into a vector. Examples of vectors usedfor such purposes include expression plasmids capable of directing theexpression of the nucleic acids in the target cell. In other instances,the vector is a viral vector system wherein the nucleic acids areincorporated into a viral genome that is capable of transfecting thetarget cell. In a preferred embodiment, the polynucleotides can beoperably linked to expression and control sequences that can directexpression of the gene in the desired target host cells. Thus, one canachieve expression of the nucleic acid under appropriate conditions inthe target cell.

[0240] B. Gene Delivery Systems

[0241] Viral vector systems useful in the expression of the nucleicacids include, for example, naturally occurring or recombinant viralvector systems. Depending upon the particular application, suitableviral vectors include replication competent, replication deficient, andconditionally replicating viral vectors. For example, viral vectors canbe derived from the genome of human or bovine adenoviruses, vacciniavirus, herpes virus, adeno-associated virus, minute virus of mice (MVM),HIV, sindbis virus, and retroviruses (including but not limited to Roussarcoma virus), and MoMLV. Typically, the genes of interest are insertedinto such vectors to allow packaging of the gene construct, typicallywith accompanying viral DNA, followed by infection of a sensitive hostcell and expression of the gene of interest.

[0242] As used herein, “gene delivery system” refers to any means forthe delivery of a nucleic acid of the invention to a target cell. Insome embodiments of the invention, nucleic acids are conjugated to acell receptor ligand for facilitated uptake (e.g., invagination ofcoated pits and internalization of the endosome) through an appropriatelinking moiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem.263:14621-14624 (1988); WO 92/06180). For example, nucleic acids can belinked through a polylysine moiety to asialo-oromucocid, which is aligand for the asialoglycoprotein receptor of hepatocytes.

[0243] Similarly, viral envelopes used for packaging gene constructsthat include the nucleic acids of the invention can be modified by theaddition of receptor ligands or antibodies specific for a receptor topermit receptor-mediated endocytosis into specific cells (see, e.g., WO93/20221, WO 93/14188, and WO 94/06923). In some embodiments of theinvention, the DNA constructs of the invention are linked to viralproteins, such as adenovirus particles, to facilitate endocytosis(Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). Inother embodiments, molecular conjugates of the instant invention caninclude microtubule inhibitors (WO/9406922), synthetic peptidesmimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem.269:12918-12924 (1994)), and nuclear localization signals such as SV40 Tantigen (WO93/19768).

[0244] Retroviral vectors are also useful for introducing the nucleicacids of the invention into target cells or organisms. Retroviralvectors are produced by genetically manipulating retroviruses. The viralgenome of retroviruses is RNA. Upon infection, this genomic RNA isreverse transcribed into a DNA copy which is integrated into thechromosomal DNA of transduced cells with a high degree of stability andefficiency. The integrated DNA copy is referred to as a provirus and isinherited by daughter cells as is any other gene. The wild typeretroviral genome and the proviral DNA have three genes: the gag, thepol and the env genes, which are flanked by two long terminal repeat(LTR) sequences. The gag gene encodes the internal structural(nucleocapsid) proteins; the pol gene encodes the RNA directed DNApolymerase (reverse transcriptase); and the env gene encodes viralenvelope glycoproteins. The 5′ and 3′ LTRs serve to promotetranscription and polyadenylation of virion RNAs. Adjacent to the 5′ LTRare sequences necessary for reverse transcription of the genome (thetRNA primer binding site) and for efficient encapsulation of viral RNAinto particles (the Psi site) (see, Mulligan, In: ExperimentalManipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann etal., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of theNational Academy of Sciences, U.S.A., 81:6349-6353 (1984)).

[0245] The design of retroviral vectors is well known to those ofordinary skill in the art. In brief, if the sequences necessary forencapsidation (or packaging of retroviral RNA into infectious virions)are missing from the viral genome, the result is a cis-acting defectwhich prevents encapsidation of genomic RNA. However, the resultingmutant is still capable of directing the synthesis of all virionproteins. Retroviral genomes from which these sequences have beendeleted, as well as cell lines containing the mutant genome stablyintegrated into the chromosome are well known in the art and are used toconstruct retroviral vectors. Preparation of retroviral vectors andtheir uses are described in many publications including, e.g., EuropeanPatent Application EPA 0 178 220; U.S. Pat. No. 4,405,712, GilboaBiotechniques 4:504-512 (1986); Mann et al., Cell 33:153-159 (1983);Cone and Mulligan Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984);Eglitis et al. Biotechniques 6:608-614 (1988); Miller et al.Biotechniques 7:981-990 (1989); Miller (1992) supra; Mulligan (1993),supra; and WO 92/07943.

[0246] The retroviral vector particles are prepared by recombinantlyinserting the desired nucleotide sequence into a retrovirus vector andpackaging the vector with retroviral capsid proteins by use of apackaging cell line. The resultant retroviral vector particle isincapable of replication in the host cell but is capable of integratinginto the host cell genome as a proviral sequence containing the desirednucleotide sequence. As a result, the patient is capable of producing,for example, a polypeptide or polynucleotide of the invention and thusrestore the cells to a normal phenotype.

[0247] Packaging cell lines that are used to prepare the retroviralvector particles are typically recombinant mammalian tissue culture celllines that produce the necessary viral structural proteins required forpackaging, but which are incapable of producing infectious virions. Thedefective retroviral vectors that are used, on the other hand, lackthese structural genes but encode the remaining proteins necessary forpackaging. To prepare a packaging cell line, one can construct aninfectious clone of a desired retrovirus in which the packaging site hasbeen deleted. Cells comprising this construct will express allstructural viral proteins, but the introduced DNA will be incapable ofbeing packaged. Alternatively, packaging cell lines can be produced bytransforming a cell line with one or more expression plasmids encodingthe appropriate core and envelope proteins. In these cells, the gag,pol, and env genes can be derived from the same or differentretroviruses.

[0248] A number of packaging cell lines suitable for the presentinvention are also available in the prior art. Examples of these celllines include Crip, GPE86, PA317 and PG13 (see Miller et al., J. Virol.65:2220-2224 (1991)). Examples of other packaging cell lines aredescribed in Cone and Mulligan Proceedings of the National Academy ofSciences, USA, 81:6349-6353 (1984); Danos and Mulligan Proceedings ofthe National Academy of Sciences, USA, 85:6460-6464 (1988); Eglitis etal. (1988), supra; and Miller (1990), supra.

[0249] Packaging cell lines capable of producing retroviral vectorparticles with chimeric envelope proteins may be used. Alternatively,amphotropic or xenotropic envelope proteins, such as those produced byPA317 and GPX packaging cell lines may be used to package the retroviralvectors.

[0250] In some embodiments of the invention, an antisense polynucleotideis administered which hybridizes to a gene encoding a polypeptide of theinvention. The antisense polypeptide can be provided as an antisenseoligonucleotide (see, e.g., Murayama et al., Antisense Nucleic Acid DrugDev. 7:109-114 (1997)). Genes encoding an antisense nucleic acid canalso be provided; such genes can be introduced into cells by methodsknown to those of skill in the art. For example, one can introduce anantisense nucleotide sequence in a viral vector, such as, for example,in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173(1997)), in adeno-associated virus (see, e.g., Xiao et al., Brain Res.756:76-83 (1997)), or in other systems including, but not limited, to anHVJ (Sendai virus)—liposome gene delivery system (see, e.g., Kaneda etal., Ann. NY Acad. Sci. 811:299-308 (1997)), a “peptide vector” (see,e.g., Vidal et al., CR Acad. Sci III 32:279-287 (1997)), as a gene in anepisomal or plasmid vector (see, e.g., Cooper et al., Proc. Natl. Acad.Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584(1997)), as a gene in a peptide-DNA aggregate (see, e.g., Niidome etal., J. Biol. Chem. 272:15307-15312 (1997)), as “naked DNA” (see, e.g.,U.S. Pat. Nos. 5,580,859 and 5,589,466), in lipidic vector systems (see,e.g., Lee et al., Crit Rev Ther Drug Carrier Syst. 14:173-206 (1997)),polymer coated liposomes (U.S. Pat. Nos. 5,213,804 and 5,013,556),cationic liposomes (Epand et al., U.S. Pat. Nos. 5,283,185; 5,578,475;5,279,833; and 5,334,761), gas filled microspheres (U.S. Pat. No.5,542,935), ligand-targeted encapsulated macromolecules (U.S. Pat. Nos.5,108,921; 5,521,291; 5,554,386; and 5,166,320). In another embodiment,conditional expression systems, such as those typified by thetet-regulated systems and the RU-486 system, can be used (see, e.g.,Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther.5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering etal., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol.16:757-761 (1998)). These systems impart small molecule control on theexpression of the target gene(s) of interest.

[0251] C. Pharmaceutical Formulations

[0252] When used for pharmaceutical purposes, the vectors used for genetherapy are formulated in a suitable buffer, which can be anypharmaceutically acceptable buffer, such as phosphate buffered saline orsodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterilewater, and other buffers known to the ordinarily skilled artisan such asthose described by Good et al. Biochemistry 5:467 (1966).

[0253] The compositions can additionally include a stabilizer, enhancer,or other pharmaceutically acceptable carriers or vehicles. Apharmaceutically acceptable carrier can contain a physiologicallyacceptable compound that acts, for example, to stabilize the nucleicacids of the invention and any associated vector. A physiologicallyacceptable compound can include, for example, carbohydrates, such asglucose, sucrose or dextrans; antioxidants, such as ascorbic acid orglutathione; chelating agents; low molecular weight proteins or otherstabilizers or excipients. Other physiologically acceptable compoundsinclude wetting agents, emulsifying agents, dispersing agents, orpreservatives, which are particularly useful for preventing the growthor action of microorganisms. Various preservatives are well known andinclude, for example, phenol and ascorbic acid. Examples of carriers,stabilizers, or adjuvants can be found in Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

[0254] D. Administration of Formulations

[0255] The formulations of the invention can be delivered to any tissueor organ using any delivery method known to the ordinarily skilledartisan. In some embodiments of the invention, the nucleic acids of theinvention are formulated in mucosal, topical, and/or buccalformulations, particularly mucoadhesive gel and topical gelformulations. Exemplary permeation enhancing compositions, polymermatrices, and mucoadhesive gel preparations for transdermal delivery aredisclosed in U.S. Pat. No. 5,346,701.

[0256] E. Methods of Treatment

[0257] The gene therapy formulations of the invention are typicallyadministered to a cell. The cell can be provided as part of a tissue,such as an epithelial membrane, or as an isolated cell, such as intissue culture. The cell can be provided in vivo, ex vivo, or in vitro.

[0258] The formulations can be introduced into the tissue of interest invivo or ex vivo by a variety of methods. In some embodiments of theinvention, the nucleic acids of the invention are introduced into cellsby such methods as microinjection, calcium phosphate precipitation,liposome fusion, or biolistics. In further embodiments, the nucleicacids are taken up directly by the tissue of interest.

[0259] In some embodiments of the invention, the nucleic acids of theinvention are administered ex vivo to cells or tissues explanted from apatient, then returned to the patient. Examples of ex vivoadministration of therapeutic gene constructs include Nolta et al., ProcNatl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars inOncology 23 (1):46-65 (1996); Raper et al., Annals of Surgery223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg.,11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA93(1):402-6 (1996).

[0260] X. Diagnosis of Mood Disorders and Psychotic Disorders

[0261] The present invention also provides methods of diagnosing mooddisorders (such as major depression or bipolar disorder), psychoticdisorders (such as schizophrenia) Diagnosis involves determining thelevel of a polypeptide or polynucleotide of the invention in a patientand then comparing the level to a baseline or range. Typically, thebaseline value is representative of a polypeptide or polynucleotide ofthe invention in a healthy person not suffering from a mood disorder orpsychotic disroder or under the effects of medication or other drugs.Variation of levels of a polypeptide or polynucleotide of the inventionfrom the baseline range (either up or down) indicates that the patienthas a mood disorder or psychotic disorder or at risk of developing atleast some aspects of a mood disorder or psychotic disorder. In someembodiments, the level of a polypeptide or polynucleotide of theinvention are measured by taking a blood, urine or tissue sample from apatient and measuring the amount of a polypeptide or polynucleotide ofthe invention in the sample using any number of detection methods, suchas those discussed herein, e.g., SNPs or haplotypes associated with thisgenes.

[0262] In some embodiments, the level of the enzymatic product of apolypeptide or polynucleotide of the invention is measured and comparedto a baseline value of a healthy person or persons. Modulated levels ofthe product compared to the baseline indicates that the patient has amood disorder or psychotic disorder or is at risk of developing at leastsome aspects of a mood disorder or psychotic disorder. Patient samples,for example, can be blood, saliva, CSF, urine or tissue samples.

[0263] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

EXAMPLES Example 1

[0264] Identification of Genes Dysregulated in Mood Disorders

[0265] A total of twenty mood disorder brains (9 bipolar and 11 majordepression disorder patients) with twenty control brains were used inthis study. Each brain pair (case and control) was matched on the basisof gender, age, and postmortem interval. Three brain regions,dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (AnCg)and the cerebellum (CB) were extracted for RNA and subjected tomicroarray analysis using Affymetrix oligonucleotide GeneChips™. EachRNA sample was subjected to two independent analyses. The results wereanalyzed using multiple statistical tools and algorithms with variousstringencies. Real time PCR analysis was used to confirm differentialgene expression for selected genes. The genes identified using thisstudy are listed in Tables 1, 2, and 3. Furthermore, biochemicalpathways associated with the differentially expressed genes wereidentified (see FIGS. 1-5).

[0266] The two cortical regions DLPFC and AnCg had similar geneexpression profiles in controls but differed significantly in MDD andBP, demonstrating distinct gene expression profiles. BP subject showedmore changes in AnCg compared to DLPFC whereas MDD show less profoundchanges in both cortical regions but had greater effects in the DLPFCthan in the AnCg. For BP, several candidate genes were located inchromosomal region 15q11-13, which is associated with the Prader-Willisyndrome (see FIGS. 6-8).

Example 2

[0267] Identification of Additional Genes Dysregulated in Mood Disorders

[0268] The RNA from three brain regions, dorsolateral prefrontal cortex(DLPFC), anterior cingulate cortex (AnCg) and the cerebellum (CB) fromdeceased patients diagnosed with bipolar disease or major depression,and matched controls were extracted and subjected to microarray analysisusing Affymetrix oligonucleotide GeneChips™. The patient's particularconditions in their terminal phase (agonal factors, e.g., seizure, coma,hypoxia, dehydration, and pyrexia) and the conditions of the braintissue after death (postmortem factors, e.g., postmortem interval, andfreezer interval) are two major influences on RNA preservation inpostmortem brain tissue. Brain pH has been evaluated as an indicator foragonal status, and as an indicator of RNA preservation. The effect ofagonal factors and pH were taken into account for quality control of theRNA. Two RNA samples were subjected to independent analyses. The resultswere analyzed using multiple statistical tools and algorithms withvarious stringencies. The 967 genes identified using this study arelisted in Table 4. Real time PCR analysis was used to confirmdifferential gene expression for selected genes. Real time PCRconfirmation of differential gene expression for selected genes islisted in Table 5.

[0269] Furthermore, biochemical pathways associated with thedifferentially expressed genes were identified. In particular, corticalareas in BP patients showed activation of several pathways, includingthe proteasome pathway, the oxidative phospharylation pathway, the ATPsynthesis pathway, and chaperones (i.e., heat shock proteins). Inaddition, signaling pathways dysregulated in BP include, e.g., G-coupledprotein receptors, the phosphatidylinositol pathway, the cAMP pathway,the mitogen activated protein kinase pathway, cytoskeletal systems, andthe cortical GABA and glutamate systems. In MD, dysregulated genesincludes genes involved in transmission of nerve impulses, neurogenesis,and the fibroblast growth factor system (FGF). (see FIGS. 10-12). Geneontology (i.e., genetic signatures) for BP and MD can conveniently beused in developing diagnostic and therapeutic regiments for mooddisorders.

Example 3

[0270] Identification of Additional Genes Dysregulated in Mood DisordersUsing Rat Models of Depression and Anti-Depressant Treatment

[0271] Rats were exposed to chronic unpredictable stress treatments inparallel with chronic anti-depressants treatment (e.g., the tricyclicantidepressant desipramine and the specific serotonin reuptake inhibitorfluoxetine). Saline treated stressed rats (SS) and saline treatednon-stressed rats (SN) were used as controls. In particular, salinetreated stressed rats (SS) were compared to desipramine treated stressedrats (DS); saline treated stressed rats (SS) were compared to fluoxetinetreated stressed rats (FS); saline treated non-stressed rats (SN) werecompared to desipramine treated non-stressed rats (DN); saline treatednon-stressed rats (SN) were compared to fluoxetine treated non-stressedrats (FN); and saline treated stressed rats (SS) were compared to salinetreated non-stressed rats (SN). Gene expression changes in rat cortexfollowing treatment were measured. The genes identified in this studyare shown in Table 6. This data suggests that different classes ofantidepressants, i.e., antidepressants with apparently differentmechanisms of action may act through a common biochemical pathway.

[0272] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences, GOterms, patents, and patent applications cited herein are herebyincorporated by reference.

What is claimed is:
 1. A method for determining whether a subject has oris predisposed for a mood disorder, the method comprising the steps of:(i) obtaining a biological sample from a subject; (ii) contacting thesample with a reagent that selectively associates with a polynucleotideor polypeptide encoded by a nucleic acid that hybridizes under stringentconditions to a nucleotide sequence of Table 2, 3, or 4; and (iii)detecting the level of reagent that selectively associates with thesample, thereby determining whether the subject has or is predisposedfor a mood disorder.
 2. The method of claim 1, wherein the reagent is anantibody.
 3. The method of claim 1, wherein the reagent is a nucleicacid.
 4. The method of claim 1, wherein the reagent associates with apolynucleotide.
 5. The method of claim 1, wherein the regent associateswith a polypeptide.
 6. The method of claim 1, wherein the level ofreagent that associates with the sample is different from a levelassociated with humans without a mood disorder.
 7. The method of claim1, wherein the biological sample is obtained from amniotic fluid.
 8. Themethod of claim 1, wherein the mood disorder is selected from the groupconsisting of bipolar disorder I, bipolar disorder II, and majordepression disorder.
 9. The method of claim 6, wherein the level ofreagent that associates with the sample is higher than a levelassociated with humans without a mood disorder.
 10. The method of claim6, wherein the level of reagent that associates with the sample is lowerthan a level associated with humans without a mood disorder.
 11. Amethod of identifying a compound for treatment or prevention of a mooddisorder, the method comprising the steps of: (i) contacting thecompound with a polypeptide, the polypeptide encoded by a polynucleotidethat hybridizes under stringent conditions to a nucleic acid sequencecomprising a nucleotide sequence of Table 2, 3, or 4; and (ii)determining the functional effect of the compound upon the polypeptide,thereby identifying a compound for treatment or prevention of a mooddisorder.
 12. The method of claim 11, wherein the contacting step isperformed in vitro.
 13. The method of claim 11, wherein the polypeptideis expressed in a cell and the cell is contacted with the compound. 14.The method of claim 11, the mood disorder is selected from the groupconsisting of bipolar disorder I, bipolar disorder II, and majordepression disorder.
 15. The method of claim 11, further comprisingadministering the compound to an animal and determining the effect onthe animal.
 16. The method of claim 15, wherein the determining stepcomprises testing the animal's mental function.
 17. A method ofidentifying a compound for treatment of a mood disorder in a subject,the method comprising the steps of: (i) contacting the compound to acell, the cell comprising a polynucleotide that hybridizes understringent conditions to a nucleotide sequence of Table 2, 3, or 4; and(ii) selecting a compound that modulates expression of thepolynucleotide, thereby identifying a compound for treatment of a mooddisorder.
 18. The method of claim 17, wherein the expression of thepolynucleotide is enhanced.
 19. The method of claim 17, wherein theexpression of the polynucleotide is decreased.
 20. The method of claim17, further comprising administering the compound to an animal anddetermining the effect on the animal.
 21. The method of claim 20,wherein the determining step comprises testing the animal's mentalfunction.
 22. The method of claim 17, wherein the mood disorder isselected from the group consisting of bipolar disorder I, bipolardisorder II, and major depression disorder.
 23. A method of treating amood disorder in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of acompound identified using the method of claim 11 or claim
 17. 24. Themethod of claim 23, wherein the mood disorder is selected from the groupconsisting of bipolar disorder I, bipolar disorder II, and majordepression disorder.
 25. The method of claim 23, wherein the compound isa small organic molecule.
 26. A method of treating a mood disorder in asubject, the method comprising the step of administering to the subjecta therapeutically effective amount of a polypeptide, the polypeptideencoded by a polynucleotide that hybridizes under stringent conditionsto a nucleotide sequence of Table 2, 3, or
 4. 27. The method of claim26, wherein the mood disorder is selected from the group consisting ofbipolar disorder I, bipolar disorder II, and major depression disorder.28. A method of treating a mood disorder in a subject, the methodcomprising the step of administering to the subject a therapeuticallyeffective amount of a nucleic acid, wherein the nucleic acid hybridizesunder stringent conditions to a nucleotide sequence of Table 2, 3, or 4.29. The method of claim 28, wherein the mood disorder is selected fromthe group consisting of bipolar disorder I, bipolar disorder II, andmajor depression disorder.